Murf gene and enzyme of Pseudomonas aeruginosa

ABSTRACT

This invention provides isolated polynucleotides that encode the MurF (UDP-N-acetylmuramyl-L-alanine-D-glutamate-m-DaD:D-alanine-D-alanine ligase) protein of  Pseudomonas aeruginosa . Purified and isolated MurF recombinant proteins are also provided. Nucleic acid sequences which encode functionally active MurF proteins are described. Assays for the identification of modulators of the expression of murF and inhibitors of the activity of MurF, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of provisional 60/153,293 filed Sep. 10,1999

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

FIELD OF THE INVENTION

This invention relates to the genes and enzymes involved in cell wallsynthesis in bacteria, and particularly to the inhibition of suchenzymes.

BACKGROUND OF THE INVENTION

The pathway of peptidoglycan biosynthesis is both essential and uniqueto bacteria, making it an attractive target for antibiotic research.Several enzymes in this pathway are molecular targets of naturallyoccurring antibiotics such as fosfomycin, cycloserine, b-lactams andvancomycin.

The construction of the peptidoglycan begins in the cytoplasm with anactivated sugar molecule, UDP-N-acetylglucosamine. After two reactions(catalyzed by MurA and MurB) that result in the placement of a lactylgroup on the 3—OH of the glucosamine moiety, a series of ATP-dependentamino acid ligases (MurC, -D, -E, and —F) catalyze the stepwisesynthesis of the pentapeptide sidechain using the newly synthesizedlactyl carboxylate as the first acceptor site. After attachment of thesugar pentapeptide to a lipid carrier in the plasma membrane, anotherglucosamine unit is added to the 4—OH of the muramic acid moiety. Thecompleted monomeric building block is moved across the membrane into theperiplasm where the penicillin-binding proteins enzymatically add itinto the growing cell wall (Lugtenberg, E. J., 1972, Studies onEscherichia coli enzymes involved in the synthesis of UridineDiphosphate-N-Acetyl-Muramyl-pentapeptide. J. Bacteriol. 110:2634;Mengin-Lecreulx, D., B. Flouret, and J. van Heijenoort, 1982,Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli. J.Bacteriol. 151: 1109-1117).

Because the pentapeptide sidechain is not synthesized ribosomally itcontains more diverse chemical functionality than a typical peptide,both structurally and stereochemically. Two of the enzymes catalyze theaddition of D-amino acids (MurD and MurF) and MurE mediates theformation of a peptide bond between the g-carboxylate of D-glutamate andthe amino group of L-lysine. Presumably these structures render theexposed peptidoglycan resistant to the action of proteases, but theyalso imply that the active sites of the enzymes must have unusualstructures in order to handle the somewhat uncommon substrates. Theseunusual active sites are targets to bind novel inhibitors that can haveantimicrobial activity.

Although peptidoglycan assembly is a proven target for antibiotics,there are no known inhibitors for many of the enzymes of the pathway.Since these enzymes are conserved among eubacteria, inhibitors of thispathway are likely to be broad spectrum antibiotics. Among thesepotential enzyme targets is MurF,UDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap: D-alanine-D-alanineligase. This enzyme is a target for the antibiotic cycloserine(Kleinkauf H and H. von Dohren. 1990. Nonribosomal biosynthesis ofpeptide antibiotics. Eur J. Biochem. 192:1-15). This validates theassumption that inhibitors of this enzyme are likely to lead toantibiotics for treating infections with either Gram (−ve) or Gram (+ve)bacteria.

SUMMARY OF THE INVENTION

Polynucleotides and polypeptides of Pseudomonas aeruginosa MurF, anenzyme involved in bacterial cell wall biosynthesis are provided. Therecombinant MurF enzyme is catalytically active in ATP-dependentD-alanine-D-alanine addition reactions. The enzyme is used in in vitroassays to screen for antibacterial compounds that target cell wallbiosynthesis. The invention includes the polynucleotides, proteinsencoded by the polynucleotides, and host cells expressing therecombinant enzyme, probes and primers, and the use of these moleculesin assays.

An aspect of this invention is a polynucleotide having a sequenceencoding a Pseudomonas aeruginosa MurF protein, or a complementarysequence. In a particular embodiment the encoded protein has a sequencecorresponding to SEQ ID NO:2. In other embodiments, the encoded proteincan be a naturally occurring mutant or polymorphic form of the protein.In preferred embodiments the polynucleotide can be DNA, RNA or a mixtureof both, and can be single or double stranded. In particularembodiments, the polynucelotide is comprised of natural, non-natural ormodified nucleotides. In some embodiments, the internucleotide linkagesare linkages that occur in nature. In other embodiments, theinternucleotide linkages can be non-natural linkages or a mixture ofnatural and non-natural linkages. In a most preferred embodiment, thepolynucleotide has a sequence shown in SEQ ID NO:1.

An aspect of this invention is a polynucleotide having a sequence of atleast about 25 contiguous nucleotides that is specific for a naturallyoccurring polynucleotide encoding a Pseudomonas aeruginosa MurF protein.In particular preferred embodiments, the polynucleotides of this aspectare useful as probes for the specific detection of the presence of apolynucleotide encoding a Pseudomonas aeruginosa MurF protein. In otherparticular embodiments, the polynucleotides of this aspect are useful asprimers for use in nucleic acid amplification based assays for thespecific detection of the presence of a polynucleotide encoding aPseudomonas aeruginosa MurF protein. In preferred embodiments, thepolynucleotides of this aspect can have additional components including,but not limited to, compounds, isotopes, proteins or sequences for thedetection of the probe or primer.

An aspect of this invention is an expression vector including apolynucleotide encoding a Pseudomonas aeruginosa MurF protein, or acomplementary sequence, and regulatory regions. In a particularembodiment the encoded protein has a sequence corresponding to SEQ IDNO:2. In particular embodiments, the vector can have any of a variety ofregulatory regions known and used in the art as appropriate for thetypes of host cells the vector can be used in. In a most preferredembodiment, the vector has regulatory regions appropriate for theexpression of the encoded protein in gram-negative prokaryotic hostcells. In other embodiments, the vector has regulatory regionsappropriate for expression of the encoded protein in gram-positive hostcells, yeasts, cyanobacteria or actinomycetes. In some preferredembodiments the regulatory regions provide for inducible expressionwhile in other preferred embodiments the regulatory regions provide forconstitutive expression. Finally, according to this aspect, theexpression vector can be derived from a plasmid, phage, virus or acombination thereof.

An aspect of this invention is host cell comprising an expression vectorincluding a polynucleotide encoding a Pseudomonas aeruginosa MurFprotein, or a complementary sequence, and regulatory regions. In aparticular embodiment the encoded protein has a sequence correspondingto SEQ ID NO:2. In preferred embodiments, the host cell is a yeast,gram-positive bacterium, cyanobacterium or actinomycete. In a mostpreferred embodiment, the host cell is a gram-negative bacterium.

An aspect of this invention is a process for expressing a MurF proteinof P. aeruginosa in a host cell. In this aspect a host cell istransformed or transfected with an expression vector including apolynucleotide encoding a Pseudomonas aeruginosa MurF protein, or acomplementary sequence. According to this aspect, the host cell iscultured under conditions conducive to the expression of the encodedMurF protein. In particular embodiments the expression is inducible orconstitutive. In a particular embodiment the encoded protein has asequence corresponding to SEQ ID NO:2.

An aspect of this invention is a purified polypeptide having an aminoacid sequence of SEQ ID NO:2 or the sequence of a naturally occurringmutant or polymorphic form of the protein.

An aspect of this invention is a method of determining whether acandidate compound can inhibit the activity of a P. aeruginosa MurFpolypeptide. According to this aspect a polynucleotide encoding thepolypeptide is used to construct an expression vector appropriate for aparticular host cell. The host cell is transformed or transfected withthe expression vector and cultured under conditions conducive to theexpression of the MurF polypeptide. The cell is contacted with thecandidate. Finally, one measures the activity of the MurF polypeptide inthe presence of the candidate. If the activity is lower relative to theactivity of the protein in the absence of the candidate, then thecandidate is a inhibitor of the MurF polypeptide. In preferredembodiments, the polynucleotide encodes a protein having an amino acidsequence of SEQ ID NO:2 or a naturally occurring mutant of polymorphicform thereof. In other preferred embodiments, the polynucleotide has thesequence of SEQ ID NO:1. In particular embodiments, the relativeactivity of MurF is determined by comparing the activity of the MurF ina host cell. In some embodiments, the host cell is disrupted and thecandidate is contacted to the released cytosol. In other embodiments,the cells can be disrupted contacting with the candidate and beforedetermining the activity of the MurF protein. Finally, according to thisaspect the relative activity can determined by comparison to apreviously measured or expected activity value for the MurF activity inthe host under the conditions. However, in preferred embodiments, therelative activity is determined by measuring the activity of the MurF ina control cell that was not contacted with a candidate compound. Inparticular embodiments, the host cell is a pseudomonad and the proteininhibited is the MurF produced by the pseudomonad.

An aspect of this invention is a compound that is an inhibitor of a P.aeruginosa MurF protein an assay described herein. In preferredembodiments, the compound is an inhibitor of a P. aeruginosa MurFprotein produced by a host cell comprising an expression vector of thisinvention. In most preferred embodiments, the compound is also aninhibitor of MurF protein produced by a pathogenic strain P. aeruginosaand also inhibits the growth of said pseudomonad.

An aspect of this invention is a pharmaceutical preparation thatincludes an inhibitor of P. aeruginosa MurF and a pharmaceuticallyacceptable carrier.

An aspect of this invention is a method of treatment comprisingadministering a inhibitor of the P. aeruginosa MurF to a patient. Thetreatment can be prophylactic or therapeutic. In preferred embodiments,the appropriate dosage for a particular patient is determined by aphysician.

By “about” it is meant within approximately 10-20% greater or lesserthan particularly stated.

As used herein an “inhibit r” is a compound that interacts with andinhibits or prevents a polypeptide of MurF from catalyzing theATP-dependent addition of D-alanine-D-alanine to an m-Dap residue of theUDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap.

As used herein a “modulator” is a compound that interacts with an aspectof cellular biochemistry to effect an increase or decrease in the amountof a polypeptide of MurF present in, at the surface or in the periplasmof a cell, or in the surrounding serum or media. The change in amount ofthe MurF polypeptide can be mediated by the effect of a modulator on theexpression of the protein, e.g., the transcription, translation,post-translational processing, translocation or folding of the protein,or by affecting a component(s) of cellular biochemistry that directly orindirectly participates in the expression of the protein. Alternatively,a modulator can act by accelerating or decelerating the turnover of theprotein either by direct interaction with the protein or by interactingwith another component(s) of cellular biochemistry which directly orindirectly effects the change.

All of the references cited herein are incorporated by reference intheir entirety as background material.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Nucleotide sequence (SEQ ID NO:1) and the predicted aminoacid sequence (SEQ ID NO:2) of P. aeruginosa murF. The amino acidsequence (SEQ ID NO:2) is presented in three-letter code below thenucleotide sequence (nucleotides 57 to 1431 of SEQ ID NO:1).

FIG. 2. Production of MurF Protein. Lane 1, Molecular weight markers;Lane2, IPTG-induced lysate of cells (BL21(DE3)/pLysS) containing thecontrol vector pET -15b; Lane 3, uninduced cell lysate containing thecontrol vector pET-15b; lane 4, column-purified MurF; Lane 5IPTG-induced lysate of cells expressing MurF; Lane 6, uninduced lysateof cells containing murF.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides polynucleotides and polypeptides of a cell wallbiosynthesis gene from Pseudomonas aeruginosa, referred to herein asMurF. The polynucleotides and polypeptides are used to further provideexpression vectors, host cells comprising the vectors, probes andprimers, antibodies against the MurF protein and polypeptides thereof,assays for the presence or expression of MurF and assays for theidentification of modulators and inhibitors of MurF.

Bacterial MurF,UDP-N-acetylmuramyl-L-alanine-D-glutamate-m-Dap:D-alanine-D-alanineligase, a cytoplasmic peptidoglycan biosynthetic enzyme, catalyzes theATP-dependent addition of D-alanine-D-alanine to the m-Dap residue ofthe UDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap precursor generatingthe pentapeptideUDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap-D-alanine-D-alanine.

N The murF gene was cloned from Pseudomonas aeruginosa. Sequenceanalysis of the P. aeruginosa murF gene revealed an open reading frameof 458 amino acids. The deduced amino acid sequence of P. aeruginosaMurF is homologous to MurF from Escherichia coli, Bacillus subtilis andother bacteria. Recombinant MurF protein from P. aeruginosa wasover-produced as His-tagged fusion protein in Escherichia coli hostcells and the enzyme was purified to apparent homogeneity. Therecombinant enzyme catalyzed the ATP-dependent addition ofD-alanine-D-alanine to theUDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap precursor.

Nucleic acids encoding murF from Pseudomonas aeruginosa are useful inthe expression and production of the P. aeruginosa MurF protein. Thenucleic acids are also useful in providing probes for detecting thepresence of P. aeruginosa murF.

Polynucleotides

Polynucleotides useful in the present invention include those describedherein and those that one of skill in the art will be able to derivetherefrom following the teachings of this specification. A preferredaspect of the present invention is an isolated nucleic acid encoding aMurF protein of Pseudomonas aeruginosa. A preferred embodiment is anucleic acid having the sequence disclosed in FIG. 1, SEQ ID NO:1 anddisclosed as follows:

-   TCCGTTCTCC GACATCGAGC AGGCCGAGCG CGCCCTGGCC GCCTGGGAGG-   TGCCGATCG TTGAGCCTCT TCGCCTCAGC CAGTTGACGG TCGCGCTGGA-   CGCCCGCCTG ATCGGCGAGG ACGCCGTCTT TTCGGCGGTT TCCACCGACA-   GTCGCGCCAT CGGGCCCGGC CAACTGTTCA TTGCCCTGAG TGGGCCGCGT-   TTCGACGGCC ACGACTATCT CGCCGAGGTT GCCGCCAAGG GCGCGGTGGC-   TGCGCTGGTG GAGCGCGAAG TCGCCGACGC GCCCTTGCCG CAATTGCTGG-   TGCGCGATAC CCGTGCGGCC CTGGGGCGAC TGGGCGCGCT GAACCGGCGC-   AAGTTCACCG GCCCGCTGGC GGCCATGACG GGCTCCAGCG GCAAGACCGC-   GGTCAAGGAG ATGCTCGCCA GCATCCTGCG TACCCAGGCC GGCGATGCCG-   AGTCGGTGCT GGCTACCCGT GGCAATCTGA ACAACGACCT CGGCGTACCG-   CTGACCCTGC TGCAACTGGC GCCGCAGCAC CGTAGCGCAG TGATCGAACT-   CGCACGTGGC GATCATCACC AATGCCGGAA CCGCCCATGT CGGCGAGTTC-   GGCGGACCGG AGAAGATCGT CGAGGCGAAG GGCGAGATAC TCGAAGGGCT-   GGCCGCCGAC GGCACCGCCG TACTGAACCT GGACGACAAG GCCTTCGACA-   CCTGGAAGGC CCGTGCCAGC GGCCGTCCGT TGCTGACTTT CTCCCTCGAC-   CGGCCCCAGG CCGATTTCCG CGCCGCCGAT CTGCAGCGCG ATGCGCGCGG-   CTGCATGGGC TTCAGGCTGC AGGGCGTAGC GGGTGAAGCG CAGGTCCAGC-   TCAACCTGCT GGGGCGGCAC AATGTCGCCA ATGCCCTGGC TGCGGCCGCT-   GCCGCCCATG CACTGGGCGT GCCGCTGGAT GGGATCGTCG CCGGGCTGCA-   GGCGCTGCAG CCGGTCAAGG GCCGCGCGGT AGCGCAACTG ACCGCCAGCG-   GGCTGCGTGT GATAGACGAC AGCTACAACG CCAACCCCGC GTCAATGCTG-   GCGGCGATTG ATATACTGAG CGGCTTTTCC GGGCGCACCG TCCTGGTCCT-   CGGAGACATG GGCGAACTCG GTTCCTGGGC CGAGCAGGCC CACCGCGAGG-   TGGGCGCCTA CGCCGCTGGC AAGGTGTCCG CGCTCTATGC GGTCGGACCG-   CTGATGGCCC ACGCCGTACA GGCGTTCGGC GCCACGGGCC GGCACTTCGC-   CGACCAGGCC AGCCTGATCG GGGCGCTGGC CACCGAACAA CCGACAACCA-   CCATTTTGAT CAAGGGTTCC CGCAGTGCGG CGATGGACAA AGTCGTCGCG-   GCGCTGTGCG GTTCCTCCGA GGAGAGTCAC TATGCTCCT GCTGCTGGC (SEQ ID NO:1)

The translation initiation and termination codons are underlined.

The isolated nucleic acid molecule of the present invention can includea ribonucleic or deoxyribonucleic acid molecule, which can be single(coding or noncoding strand) or double stranded, as well as syntheticnucleic acid, such as a synthesized, single stranded polynucleotide.

The present invention also relates to recombinant vectors andrecombinant hosts, both prokaryotic and eukaryotic, which contain thesubstantially purified nucleic acid molecules disclosed throughout thisspecification.

As used herein a “polynucleotide” is a nucleic acid of more than onenucleotide. A polynucleotide can be made up of multiple polynucleotideunits that are referred to by description of the unit. For example, apolynucleotide can comprise within its bounds a polynucleotide(s) havinga coding sequence(s), a polynucleotide(s) that is a regulatory region(s)and/or other polynucleotide units commonly used in the art.

An “expression vector” is a polynucleotide having regulatory regionsoperably linked to a coding region such that, when in a host cell, theregulatory regions can direct the expression of the coding sequence. Theuse of expression vectors is well known in the art. Expression vectorscan be used in a variety of host cells and, therefore, the regulatoryregions are preferably chosen as appropriate for the particular hostcell.

A “regulatory region” is a polynucleotide that can promote or enhancethe initiation or termination of transcription or translation of acoding sequence. A regulatory region includes a sequence that isrecognized by the RNA polymerase, ribosome, or associated transcriptionor translation initiation or termination factors of a host cell.Regulatory regions that direct the initiation of transcription ortranslation can direct constitutive or inducible expression of a codingsequence.

Polynucleotides of this invention contain full length or partial lengthsequences of the MurF gene sequences disclosed herein. Polynucleotidesof this invention can be single or double stranded. If single stranded,the polynucleotides can be a coding, “sense,” strand or a complementary,“antisense,” strand. Antisense strands can be useful as modulators ofthe gene by interacting with RNA encoding the MurF protein. Antisensestrands are preferably less than full length strands having sequencesunique or specific for RNA encoding the protein.

The polynucleotides can include deoxyribonucleotides, ribonucleotides ormixtures of both. The polynucleotides can be produced by cells, incell-free biochemical reactions or through chemical synthesis.Non-natural or modified nucleotides, including inosine, methylcytosine,deaza-guanosine, etc., can be present. Natural phosphodiesterinternucleotide linkages can be appropriate. However, polynucleotidescan have non-natural linkages between the nucleotides. Non-naturallinkages are well known in the art and include, without limitation,methylphosphonates, phosphorothioates, phosphorodithioates,phosphoroamidites and phosphate ester linkages. Dephospho-linkages arealso known, as bridges between nucleotides. Examples of these includesiloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, andthioether bridges. “Plastic DNA,” having, for example, N-vinyl,methacryloxyethyl, methacryamide or ethyleneimine internucleotidelinkages, can be used. “Peptide Nucleic Acid” (PNA) is also useful andresists degradation by nucleases. These linkages can be mixed in apolynucleotide.

As used herein, “purified” and “isolated” are utilized interchangeablyto stand for the proposition that the polynucleotide, protein andpolypeptide, or respective fragments thereof in question have beenremoved from the in vivo environment so that they exist in a form orpurity not found in nature. Purified or isolated nucleic acid moleculescan be manipulated by the skilled artisan, such as but not limited tosequencing, restriction digestion, site-directed mutagenesis, andsubcloning into expression vectors for a nucleic acid fragment as wellas obtaining the wholly or partially purified protein or proteinfragment so as to afford the opportunity to generate polyclonalantibodies, monoclonal antibodies, or perform amino acid sequencing orpeptide digestion. Therefore, the nucleic acids claimed herein can bepresent in whole cells or in cell lysates or in a partially orsubstantially purified form. It is preferred that the molecule bepresent at a concentration at least about five-fold to ten-fold higherthan that found in nature. A polynucleotide is considered substantiallypure if it is obtained purified from cellular components by standardmethods at a concentration of at least about 100-fold higher than thatfound in nature. A polynucleotide is considered essentially pure if itis obtained at a concentration of at least about 1000-fold higher thanthat found in nature. We most prefer polynucleotides that have beenpurified to homogeneity, that is, at least 10,000-100,000 fold. Achemically synthesized nucleic acid sequence is considered to besubstantially purified when purified from its chemical precursors by thestandards stated above.

Included in the present invention are assays that employ further novelpolynucleotides that hybridize to P.aeruginosa murf sequences understringent conditions. By way of example, and not limitation, a procedureusing conditions of high stringency is as follows: Prehybridization offilters containing DNA is carried out for 2 hr. to overnight at 65° C.in buffer composed of 6×SSC, 5× Denhardt's solution, and 100 μg/mldenatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at65° C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 37° C. for 1 hr in a solution containing 2×SSC, 0.1% SDS. Thisis followed by a wash in 0.1×SSC, 0.1% SDS at 50° C. for 45 min. beforeautoradiography.

Other procedures using conditions of high stringency would includeeither a hybridization step carried out in 5×SSC, 5× Denhardt'ssolution, 50% formamide at 42° C. for 12 to 48 hours or a washing stepcarried out in 0.2×SSPE, 0.2% SDS at 65° C. for 30 to 60 minutes.

Reagents mentioned in the foregoing procedures for carrying out highstringency hybridization are well known in the art. Details of thecomposition of these reagents can be found in, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, second edition, ColdSpring Harbor Laboratory Press. In addition to the foregoing, otherconditions of high stringency which may be used are well known in theart.

Polypeptides

A preferred aspect of the present invention is a substantially purifiedform of the MurF protein from Pseudomonas aeruginosa. A preferredembodiment is a protein that has the amino acid sequence which is shownin FIG. 1, in SEQ ID NO:2 and disclosed as follows:

-   MetLeuGluProLeuArgLeuSerGlnLeuThrValAlaLeuAspAlaArgLeuIleGly    GluAspAlaValPheSerAlaValSerThrAspSerArgAlaIeGlyProGlyGlnLeu    PheIleAlaLeuserGlyProArgPheAspGlyHisAspTyrLeuAlaGluvalAlaAla    LysGlyAlaValAlaAlaLeuValGluArgGluValAlaAspAlaProLeuProGlnLeu    LeuValArgAspThrArgAlaAlaLeuGlyArgLeuGlyAlaLeuAsnArgArgLysPhe    ThrGlyProLeuAlaAlaMetThrGlySerSerGlyLysThrAlaValLysGluMetLeu    AlaSerIleLeuArgThrGlnAlaGlyAspAlaGluSerValLeuAlaThrArgGlyAsn    LeuAsnAsnAspLeuGlyValProLeuThrLeuLeuGlnLeuAlaProGlnHisArgser    AlaValIleGluLeuGlyAlaSerArgIleGlyGluIleAlaTyrThrValGluLeuThr    ArgProHisvalAlaIlelleThrAsnAlaGlyThrAlaHisValGlyGluPheGlyGly    ProGluLysIleValGluAlaLysGlyGluIleLeuGluGlyLeuAlaAlaAspGlyThr    AlaValLeuAsnLeuAspAspLysAlaPheAspThrTrpLysAlaArgAlaSerGlyArg    ProLeuLeuThrPheSerLeuAspArgProGlnAlaAspPheArgAlaAlaAspLeuGln    ArgAspAlaArgGlyCysMetGlyPheArgLeuGlnGlyValAlaGlyGluAlaGlnVal    GlnLeuAsnLeuLeuGlyArgHisAsnValAlaAsnAlaLeuAlaAlaAlaAlaAlaAla    HiSAlaLeuGlyValProLeuAspGlylleValAlaGlyLeuGlnAlaLeuGlnProVal    LysGlyArgA laValAlaGlnLeuThrAlaSerGlyLeuArgValIleAspAspSerTyr    AsnAlaAsnProAlaSerMetLeuAlaAlaIleAspIleLeuSerGlyPheSerGlyArg    ThrValLeuValLeuGlyAspMetGlyGluLeuGlySerTrpAlaGluGlnAlaHisArg    GluValGlyAlaTyrAlaAlaGlyLysValSerAlaLeuTyrAlaValGlyProLeuMet    AlaHisAlaValGlnAlaPheGlyAlaThrGlyArgHisPheAlaAspGlnAlaSerLeu    IleGlyAlaLeuAlaThrGluGlnProThrThrThrIleLeuIleLysGlySerArgser    AlaAlaMetAspLysValValAlaAlaLeuCysGlySerSerGluGluSerHis (SEQ ID NO:2)

The present invention also relates to biologically active fragments andmutant or polymorphic forms of MurF polypeptide sequence as set forth asSEQ ID NO: 2, including but not limited to amino acid substitutions,deletions, additions, amino terminal truncations and carboxy-terminaltruncations such that these mutations provide for proteins or proteinfragments of diagnostic, therapeutic or prophylactic use and would beuseful for screening for modulators, and/or inhibitors of MurF function.

Using the disclosure of polynucleotide and polypeptide sequencesprovided herein to isolate polynucleotides encoding naturally occurringforms of MurF, one of skill in the art can determine whether suchnaturally occurring forms are mutant or polymorphic forms of MurF bysequence comparison. One can further determine whether the encodedprotein, or fragments of any MurF protein, is biologically active byroutine testing of the protein of fragment in a in vitro or in vivoassay for the biological activity of the MurF protein. For example, onecan express N-terminal or C-terminal truncations, or internal additionsor deletions, in host cells and test for their ability to catalyze theATP-dependent addition of D-alanine-D-alanine to theUDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap precursor.

It is known that there is a substantial amount of redundancy in thevarious codons which code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that encode RNAcomprising alternative codons which code for the eventual translation ofthe identical amino acid Therefore, the present invention disclosescodon redundancy which can result in different DNA molecules encoding anidentical protein. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein which donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of valine for leucine,arginine for lysine, or asparagine for glutamine may not cause a changein functionality of the polypeptide. However, any given change can beexamined for any effect on biological function by simply assaying forthe ability to catalyze the ATP-dependent addition ofD-alanine-D-alanine to theUDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap precursor as compared toan unaltered MurF protein.

It is known that DNA sequences coding for a peptide can be altered so asto code for a peptide having properties that are different than those ofthe naturally occurring peptide. Methods of altering the DNA sequencesinclude but are not limited to site directed mutagenesis. Examples ofaltered properties include but are not limited to changes in theaffinity of an enzyme for a substrate.

As used herein, a “biologically active equivalent” or “functionalderivative” of a wild-type MurF possesses a biological activity that issubstantially similar to the biological activity of a wild type MurF.The term “functional derivative” is intended to include the “fragments,”“mutants,” “variants,” “degenerate variants,” “analogs,” “orthologues,”and “homologues” and “chemical derivatives” of a wild type MurF proteinthat can catalyze the ATP-dependent addition of D-alanine-D-alanine tothe UDP-N-acetylmuramyl-L-alanine-D-Glutamine-m-Dap precursor. The term“fragment” refers to any polypeptide subset of wild-type MurF. The term“mutant” is meant to refer to a molecule that may be substantiallysimilar to the wild-type form but possesses distinguishing biologicalcharacteristics. Such altered characteristics include but are in no waylimited to altered substrate binding, altered substrate affinity andaltered sensitivity to chemical compounds affecting biological activityof the MurF or MurF functional derivative. The term “variant” refers toa molecule substantially similar in structure and function to either theentire wild-type protein or to a fragment thereof. A molecule is“substantially similar” to a wild-type MurF-like protein if bothmolecules have substantially similar structures or if both moleculespossess similar biological activity. Therefore, if the two moleculespossess substantially similar activity, they are considered to bevariants even if the exact structure of one of the molecules is notfound in the other or even if the two amino acid sequences are notidentical. The term “analog” refers to a molecule substantially similarin function to either the full-length MurF protein or to a biologicallyactive fragment thereof.

As used herein in reference to a MurF gene or encoded protein, a“polymorphic” MurF is a MurF that is naturally found in the populationof Pseudomonads at large. A polymorphic form of MurP can be encoded by adifferent nucleotide sequence from the particular murF gene disclosedherein as SEQ ID NO:1. However, because of silent mutations, apolymorphic murF gene can encode the same or different amino acidsequence as that disclosed herein. Further, some polymorphic forms MurFwill exhibit biological characteristics that distinguish the form fromwild-type MurF activity, in which case the polymorphic form is also amutant A protein or fragment thereof is considered purified or isolatedwhen it is obtained at least partially free from it's naturalenvironment in a composition or purity not found in nature. It ispreferred that the molecule be present at a concentration at least aboutfive-fold to ten-fold higher than that found in nature. A protein orfragment thereof is considered substantially pure if it is obtained at aconcentration of at least about 100-fold higher than that found innature. A protein or fragment thereof is considered essentially pure ifit is obtained at a concentration of at least about 1000-fold higherthan that found in nature. We most prefer proteins that have beenpurified to homogeneity, that is, at least 10,000-100,000 fold.

Probes and Primers

Polynucleotide probes comprising full length or partial sequences of SEQID NO:1 can be used to determine whether a cell or sample contains P.aeruginosa MurF DNA or RNA. The effect of modulators that effect thetranscription of the murF gene can be studied via the use of theseprobes. A preferred probe is a single stranded antisense probe having atleast the full length of the coding sequence of murF. It is alsopreferred to use probes that have less than the full length sequence,and contain sequences specific for P. aeruginosa murF DNA or RNA. Theidentification of a sequence(s) for user as a specific probe is wellknown in the art and involves choosing a sequence(s) that is unique tothe target sequence, or is specific thereto. It is preferred thatpolynucleotides that are probes have at least about 25 nucleotides, morepreferably about 30 to 35 nucleotides. The longer probes are believed tobe more specific for P. aeruginosa murF gene(s) and RNAs and can be usedunder more stringent hybridization conditions. Longer probes can be usedbut can be more difficult to prepare synthetically, or can result inlower yields from a synthesis. Examples of sequences that are useful asprobes or primers for P. aeruginosa murF gene(s) are Primer A (sense)5′-TTTCATATGCTTGAGCCTCTTCGCCTC-3′ (SEQ ID NO:3) and Primer B (antisense)5′-TTGGATCCTTAGTGACTCTCCTCGGAG-3′ (SEQ ID NO:4). These primers arenucleotides 1-21(A) and the complement of nucleotides 1358-1376 (B)respectively, of SEQ ID NO:1. Restriction sites, underlined, for NdeIand BamHI are added to the 5′ ends of the primers to allow cloningbetween the NdeI and BamHI sites of the expression vector pET-15b.However, one skilled in the art will recognize that these are only a fewof the useful probe or primer sequences that can be derived from SEQ IDNO:1.

Polynucleotides having sequences that are unique or specific for P.aeruginosa murF can be used as primers in amplification reaction assays.These assays can be used in tissue typing as described herein.Additionally, amplification reactions employing primers derived from P.aeruginosa murF sequences can be used to obtain amplified P. aeruginosamurF DNA using the murF DNA of the cells as an initial template. ThemurF DNA so obtained can be a mutant or polymorphic form of P.aeruginosa murF that differs from SEQ ID NO:1 by one or more nucleotidesof the murF open reading frame or sequences flanking the ORF. Thedifferences can be associated with a non-defective naturally occurringform or with a defective form of MurF. Thus, polynucleotides of thisinvention can be used in identification of various polymorphic P.aeruginosa murF genes or the detection of an organism having a P.aeruginosa murF gene. Many types of amplification reactions are known inthe art and include, without limitation, Polymerase Chain Reaction,Reverse Transcriptase Polymerase Chain Reaction, Strand DisplacementAmplification and Self-Sustained Sequence Reaction. Any of these or likereactions can be used with primers derived from SEQ ID NO:1.

Expression of MurF

A variety of expression vectors can be used to express recombinant MurFin host cells. Expression vectors are defined herein as nucleic acidsequences that include regulatory sequences for the transcription ofcloned DNA and the translation of their mRNAs in an appropriate host.Such vectors can be used to express a bacterial gene in a variety ofhosts such as bacteria, bluegreen algae, plant cells, insect cells andanimal cells. Specifically designed vectors allow the shuttling of genesbetween hosts such as bacteria-yeast or bacteria-animal cells. Anappropriately constructed expression vector should contain: an origin ofreplication for autonomous replication in host cells, selectablemarkers, a limited number of useful restriction enzyme sites, apotential for high copy number, and regulatory sequences. A promoter isdefined as a regulatory sequence that directs RNA polymerase to bind toDNA and initiate RNA synthesis. A strong promoter is one which causesmRNAs to be initiated at high frequency. Expression vectors can include,but are not limited to, cloning vectors, modified cloning vectors,specifically designed plasmids or viruses.

In particular, a variety of bacterial expression vectors can be used toexpress recombinant MurF in bacterial cells. Commercially availablebacterial expression vectors which are suitable for recombinant MurFexpression include, but are not limited to pQE (Qiagen), pET11a orpET15b (Novagen), lambda gt11 (Invitrogen), and pKK223-3 (Pharmacia).

Alternatively, one can express murF DNA in cell-freetranscription-translation systems, or murF RNA in cell-free translationsystems. Cell-free synthesis of MurF can be in batch or continuousformats known in the art.

One can also synthesize MurF chemically, although this method is notpreferred.

A variety of host cells can be employed with expression vectors tosynthesize MurF protein. These can include E. coli, Bacillus, andSalmonella. Insect and yeast cells can also be appropriate.

Following expression of MurF in a host cell, MurF polypeptides can berecovered. Several protein purification procedures are available andsuitable for use. MurF protein and polypeptides can be purified fromcell lysates and extracts, or from culture medium, by variouscombinations of, or individual application of methods includingultrafiltration, acid extraction, alcohol precipitation, saltfractionation, ionic exchange chromatography, phosphocellulosechromatography, lecithin chromatography, affinity (e.g., antibody orHis-Ni) chromatography, size exclusion chromatography, hydroxylapatiteadsorption chromatography and chromatography based on hydrophobic orhydrophillic interactions. In some instances, protein denaturation andrefolding steps can be employed. High performance liquid chromatography(HPLC) and reversed phase HPLC can also be useful. Dialysis can be usedto adjust the final buffer composition.

The MurF protein itself is useful in assays to identify compounds thatmodulate the activity of the protein—including compounds that inhibitthe activity of the protein. The MurF protein is also useful for thegeneration of antibodies against the protein, structural studies of theprotein, and structure/function relationships of the protein.

Modulators and Inhibitors of MurF

The present invention is also directed to methods for screening forcompounds which modulate or inhibit a MurF protein. Compounds whichmodulate or inhibit MurF can be DNA, RNA, peptides, proteins, ornon-proteinaceous organic or inorganic compounds or other types ofmolecules. Compounds that modulate the expression of DNA or RNA encodingMurF or are inhibitors of the biological function of MurF can bedetected by a variety of assays. The assay can be a simple “yes/no”assay to determine whether there is a change in expression or function.The assay can be made quantitative by comparing the expression orfunction of a test sample with the levels of expression or function in astandard sample, that is, a control. A compound that is a modulator canbe detected by measuring the amount of the MurF produced in the presenceof the compound. An compound that is an inhibitor can be detected bymeasuring the specific activity of the MurF protein in the presence andabsence of the compound.

The proteins, DNA molecules, RNA molecules and antibodies lendthemselves to the formulation of kits suitable for the detection andanalysis of MurF. Such a kit would comprise a compartmentalized carriersuitable to hold in close confinement at least one container. Thecarrier would further comprise reagents such as recombinant MurF oranti-MurF antibodies suitable for detecting MurP. The carrier can alsocontain a means for detection such as labeled antigen or enzymesubstrates or the like.

Pharmaceutical Compositions

Pharmaceutically useful compositions comprising a modulator or inhibitorof MurF can be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation can be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the inhibitor.

Therapeutic, prophylactic or diagnostic compositions of the inventionare administered to an individual in amounts sufficient to treat,prevent or diagnose disorders. The effective amount can vary accordingto a variety of factors such as the individual's condition, weight, sexand age. Other factors include the mode of administration. Theappropriate amount can be determined by a skilled physician

The pharmaceutical compositions can be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties can improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties canattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein can beused alone at appropriate dosages. Alternatively, co-administration orsequential administration of other agents can be desirable.

The present invention also provides a means to obtain suitable topical,oral, systemic and parenteral pharmaceutical formulations for use in themethods of treatment of the present invention. The compositionscontaining compounds identified according to this invention as theactive ingredient can be administered in a wide variety of therapeuticdosage forms in conventional vehicles for administration. For example,the compounds can be administered in such oral dosage forms as tablets,capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by injection. Likewise, they canalso be administered in intravenous (both bolus and infusion),intraperitoneal, subcutaneous, topical with or without occlusion, orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts.

Advantageously, compounds of the present invention can be administeredin a single daily dose, or the total daily dosage can be administered individed doses of two, three or four times daily. Furthermore, compoundsfor the present invention can be administered in intranasal form viatopical use of suitable intranasal vehicles, or via transdermal routes,using those forms of transdermal skin patches well known to those ofordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds of the present invention isselected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal, hepatic and cardiovascular function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

The following examples are presented by the way of illustration and,because various other embodiments will be apparent to those in the art,the following is not to be construed as a limitation on the scope of theinvention. For example, while particular preferred embodiments of theinvention are presented herein, it is within the ability of persons ofordinary skill in the art to modify or substitute vectors, host cells,compositions, etc., or to modify or design protocols or assays, all ofwhich may reach the same or equivalent performance or results as theembodiments shown herein.

EXAMPLE 1

General Materials and Methods

All reagents were purchased from SIGMA CHEMICAL CO., St. Louis, Mo.,unless otherwise indicated. UDP-N-acetylmuramyl-L-alanine wassynthesized and purified by a method known in the art (Jin, H.,Emanuele, J. J., Jr., Fairman, R., Robertson, J. G., Hail, M. E., Ho,H.-T., Falk, P. and Villafranca, J. J, 1996. Structural studies ofEscherichia coli UDP-N-acetylmuramate: L-alanine ligase, Biochemistry35: 14423-14431).

DNA Manipulations Reagents and Techniques.

Restriction endonucleases and T4 ligase were obtained from Gibco-BRL.Agarose gel electrophoresis and plasmid DNA preparations were performedaccording to published procedures (Sambrook, J., E. F. Fritsch, and T.Maniatis, 1989, Molecular cloning: a L, Laboratory Manual, 2nd ed. ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory). Recombinantplasmids containing P. aeruginosa murF were propagated in E. coli DH5a(GIBCO-BRL, Rockville, Md.) prior to protein expression in E. coliBL21(DE3)/plysS (NOVAGEN, Madison, Wis.). SDS-PAGE was performed withprecast gels (NOVAGEN). DNA sequences were determined using an automatedABI PRIS™ DNA sequencer (PERKIN-ELMER ABI, Foster City, Calif.).

EXAMPLE 2

Cloning of Pseudomonas aeruginosa murF

Genomic DNA from P. aeruginosa (strain MB4439) was prepared from 100 nmllate stationary phase culture in Brain Heart Infusion broth (DIFCO,Detroit, W). Cells were washed with 0.2 M sodium acetate, suspended in10 ml of TEG (100 mM Tris, pH 7, containing 10 mM EDTA and 25% glucose)and lysed by incubation with 200 μg of N-acetylmuramidase (SIGMA) for 1h at 37° C. Chromosomal DNA was purified from the cell lysate using aQIAGEN (Santa Clarita, Calif.) genomic DNA preparation kit and followingthe manufacturers protocol. Briefly, the cell lysate was treated withprotease K at 50° C. for 45 min, loaded onto an equilibrated QIAGENgenomic tip, entered into the resin by centrifugation at 3000 rpm for 2min. Following washing the genomic tip, the genomic DNA was eluted indistilled water and kept at 4° C. Approximately 50 ng genomic DNA wasused as a template in PCR reactions to clone murF.

Two oligonucleotide primers (GIBCO/BRL, Bethesda, Md.) complementary tosequences at the 5′ and the 3′ ends of P. aeruginosa murF were used toclone this gene using KLENTAQ ADVANTAG™ polymerase (CLONTECH, Palo Alto,Calif.). The primer nucleotide sequences were as follows:5′-TTTCATATGCTTGAGCCTCTTCGCCTC-3′ (SEQ ID NO:3) (a NdeI linker plusnucleotides 1-21 of SEQ ID NO:1) and 5′-TTGGATCCTTAGTGACTCTCCTCGGAG-3′(SEQ ID NO:4) (a BamHI linker plus the complement of nucleotides1358-1376 of SEQ ID NO:1). A PCR product representing P. aeruginosa murFwas verified by nucleotide sequence, digested with NdeI and BamHI, andcloned between the NdeI and BamHI sites of pET-I Sb, creating plasmidpPaeMurF. This plasmid was used for expression of the murF gene in E.coli.

EXAMPLE 3

Sequence Analysis of Pseudomonas aeruginosa murF

The nucleotide sequence of murF, determined in both orientations, andthe deduced amino acid sequence of the MurF protein is depicted in FIG.1. Sequence comparison using the BLAST (1) algorithm against the GenBankdatabase showed that, to varying degrees, the cloned region ishomologous (62% similar, 44% identical) to murF gene from E. coli(Parquet, C., D., Mengin-Lecreulx, B. Flouret, D. Mengin-lecreulx, andJ. van Heijenoort, 1989. Nucleotide sequence of the murF gene encodingthe UDP-MurNAc-pentapeptide synthetase of Escherichia coli., NucleicAcids Res. 17:5379).

EXAMPLE 4

Overexpression, Purification and Enzymatic Activity of Pseudomonasaeruginosa MurF

murF was cloned into the expression vector pET-15b (Novagen) asdescribed above to create plasmid pPaeMurF. The pET-15b vectorincorporates the 6× Histidine-tag into the protein construct to allowrapid purification of MurF by affinity chromatography. The pET plasmidsfor Expression by T7 RNA polymerase) plasmids are derived from pBR322and designed for protein over-production in E. coli. The vector pET-15bcontains the ampicillin resistance gene, ColE1 origin of replication inaddition to T7 phage promoter and terminator. The T7 promoter isrecognized by the phage T7 RNA polymerase but not by the E. coli RNApolymerase. A host E coli strain such as BL21(DE3)pLysS is engineered tocontain integrated copies of T7 RNA polymerase under the control oflacUV5 that is inducible by IPTG. Production of a recombinant protein inthe E. coli strain BL21(DE3)pLysS occurs after expression of T7RNApolymerase is induced.

The pPaeMurF plasmid was introduced into the host strain BL21 DE3/pLysS(NOVAGEN) for expression of His-tagged MurF. Colonies were grown at 37°C. in 100 ml of LB broth containing 100 mg/ml ampicillin and 32 μg/mlchloramphenicol. When cultures reached a cell density of A₆₀₀=0.5, cellswere pelleted and then resuspended in M9ZB medium (NOVAGEN) containing 1mM IPTG. Cells were induced for 3 h at 30° C., pelleted at 3000g, andfrozen at −80° C.

Cultures containing either the recombinant plasmid pPaeMurF or thecontrol plasmid vector, pET-15b were grown at 30° C. and induced withIPTG. Cells transformed with pPaeMurF contained an inducible protein ofapproximately 51.6 kDa, corresponding to the expected size of P.aeruginosa MurF protein as shown by SDS-PAGE. There were no comparabledetectable protein bands after induction of cells transformed with thecontrol plasmid vector, pET-15b.

Purification of Recombinant MurF Enzyme.

The cell pellet from 100 ml of induced culture prepared as describedabove was resuspended in 10 ml BT buffer (50 mM bis-tris-propane, pH8.0, containing 100 mM potassium chloride and 1% glycerol) at 4° C.Cells were lysed either by freeze-thaw or by French Press. Aftercentrifugation, the supernatant was mixed with 15 ml of freshly preparedTALON (CLONTECH) resin and incubated for 30 min at room temp. The resinwas washed twice by centrifugation with 25 ml of BT buffer at roomtemperature. Finally, the resin was loaded into a column and washed with20 ml of BT, pH 7.0, containing 5 mM imidazole. Protein was eluted with20 ml of BT buffer pH 8.0, containing 100 mM imidazole. Fractions (0.5ml) were collected and analyzed by SDS-Gel electrophoresis. Thisresulted in a partially purified preparation of P. aeruginosa MurFprotein that could be used in activity assays. The protein may bepurified further, if desired, using methods known in the art.

The P. aeruginosa murF was cloned into pET-15b between the NdeI and theBamHI sites and expressed in E. coli strain BL21(DE3)/pLysS. Therecombinant MurF enzyme was affinity purified and eluted in 100 mMimidazole. Aliquots from cell lysates, either uninduced or induced withIPTG, and column-purified polypeptides were analyzed by SDS-PAGE (FIG.2).

Assay for Activity of MurF Enzyme.

The ATP-dependent MurF activity was assayed by monitoring the formationof product ADP using the pyruvate kinase and lactate dehydrogenasecoupled enzyme assay. The reaction was monitored spectrophotometrically.

Typically, the assay contained 100 mM BIS-TRIS-propane, pH 8.0, 200 μMNADH, 1 mM ATP, 20 mM PEP, 5 MM MgCl₂, 1 mM DTT, 350 μMUDP-N-acetyl-muramyl-L-alanine-D-Glutamine-m-Dap, 1 mMD-alanine-D-alanine, 33 units/ml of pyruvate kinase and 1660 units/ml oflactate dehydrogenase in a final volume of 200 or 400 μl. The mixturewas incubated at 25° C. for 5 min and the reaction initiated by theaddition of −10 μg of MurF. These conditions are one example of an assayuseful for evaluating the activity of MurF. Other assays can be used, oramounts of buffers, substrate and enzyme can be changed, as desired, toalter the rate of production of ADP.

ADP formation was monitored by the decrease in absorbance at 340 nm as afunction of time using a SPECTRAMAXPLUS (MOLECULAR DEVICES)microtiterplate spectrophotomer (for 200 μl assays) or a HEWLETT-PACKARDHP8452A spectrophotometer equipped with a circulating water bath (for400 μl assays). Rates were calculated from the linear portions of theprogress curves using the extinction coefficient for NADH, e=6220 cm⁻¹M⁻¹. One unit of MurF activity is equal to 1 μmol of ADP formed per minat 25° C. MurF activity co-eluted with a −51 kDa protein.

TABLE 1 Specific activities of recombinant MurF from E. coli and P.aeruginosa. P. aeruginosa E. coli Mur Ligase μmol × min⁻¹ × mg⁻¹ μmol ×min⁻¹ × mg⁻¹ MurF 3.41 1.15

EXAMPLE 5

Screening for Inhibitors of MurF

One assay for the measurement of the activity of MurF is provided inExample 4. That assay, and other assays for MurF activity can be adaptedfor screening assays to detect inhibitors of MurF. For example, forinhibition assays, inhibitors in DMSO are added at the desiredconcentration to the assay mixture. In a separate, control reaction,only DMSO is added to the assay mixture. The reactions are initiated bythe addition of enzyme (MurF). Rates are calculated as described above.Relative activities are calculated from the equation 1:relative activity=rate with inhibitor/rate without inhibitor.  (1)Inhibition constant (IC₅₀) values are determined from a range ofinhibitor concentrations and calculated from equation 2.relative activity=1/(1+[I]/IC ₅₀)  (2)

One can use computer software to assist in the analysis, e.g., SIGMAPLOT™ (JANDEL SCIENTIFIC, San Rafeal).

We prefer inhibitors of MurF that result in relative activities of theMurF enzyme of at least less than 75%, more preferably, 25-50% or10-25%. We most prefer inhibitors resulting in relative activities ofless than 20%, particularly less than 10% of the activity of MurF in theabsence of the inhibitor.

We also prefer inhibitors that effectively lower the relative activityof MurF when the inhibitor is present at a very low concentration.

EXAMPLE 8

Therapy using Inhibitors of MurF

A patient presenting with an indication of infection with amicroorganism susceptible to inhibitors of MurF, e.g., gram positive andnegative bacteria, including P. aeruginosa, can be treated byadministration of inhibitors of MurF. Physicians skilled in the art arefamiliar with administering therapeutically effective amounts ofinhibitors or modulators of microbial enzymes. Such skilled persons canreadily determine an appropriate dosing scheme to achieve a desiredtherapeutic effect.

Therapy can also be prophylactic. For example, a patient at risk fordeveloping a bacterial infection, including infection with P.aeruginosa, can be treated by administration of inhibitors of MurF.Physicians skilled in the art are familiar with administeringtherapeutically effective amounts of inhibitors or modulators ofmicrobial enzymes. Such skilled persons can readily determine anappropriate dosing scheme to achieve a desired therapeutic effect.

1. A purified and isolated polynucleotide selected from the groupconsisting of: (a) a polynucleotide encoding a polypeptide having theamino acid sequence of SEQ ID NO: 2, and (b) a polynucleotide which iscomplementary to the polynucleotide of (a).
 2. The polynucleotide ofclaim 1 wherein the polynucleotide comprises nucleotides selected fromthe group consisting of natural, non-natural and modified nucleotides.3. The polynucleotide of claim 1 wherein the internucleotide linkagesare selected from the group consisting of natural and non-naturallinkages.
 4. The polynucleotide of claim 1 comprising the nucleotidesequence of SEQ ID NO:1.
 5. An expression vector comprising apolynucleotide encoding a polypeptide having the amino acid sequence ofSEQ ID NO:2.
 6. A host cell comprising the expression vector of claim 5.7. A process for expressing a MurF protein of Pseudomonas aeruginosa ina recombinant host cell, comprising: (a) transforming a suitable hostcell with an expression vector of claim 5; and (b) culturing the hostcell of step (a), under conditions which allow expression of said theMurF protein from said expression vector.
 8. A purified and isolatedpolypeptide having the amino acid sequence of SEQ ID NO:2.
 9. A methodof determining whether a candidate compound is an inhibitor of aPseudomonas aeruginosa MurF polypeptide comprising: (a) providing atleast one host cell harboring an expression vector that includes apolynucleotide encoding a polypeptide having the amino acid sequence ofSEQ ID NO: 2, (b) contacting at least one of said cells with thecandidate to permit the interaction of the candidate with the MurFpolypeptide, and (c) determining whether the candidate is an inhibitorof the MurF polypeptide by ascertaining the relative activity of thepolypeptide in the presence of the candidate.
 10. The method of claim 9wherein the polynucleotide has the nucleotide sequence of SEQ ID NO:1.11. The method of claim 9 wherein in step (c) the relative activity isdetermined by comparing a measurement of MurF polypeptide activity of atleast one cell before step (b) to a measurement of MurF polypeptideactivity of at least one cell after step (b).
 12. A method ofdetermining whether a candidate compound is an inhibitor of aPseudomonas aeruginosa MurF polypeptide comprising: (a) providing asample that includes a MurF polypeptide having the amino acid sequneceof SEQ ID NO: 2, (b) contacting said sample with the candidate to permitthe interaction of the candidate with the MurF polypeptide, and (c)determining whether the candidate is an inhibitor of the MurFpolypeptide by ascertaining the relative activity of the MurFpolypeptide in the presence of the candidate.
 13. The method of claim 12wherein in step (c) the relative activity is determined by comparing ameasurement of MurF polypeptide activity of the sample before step (b)to a measurement of MurF polypeptide activity of the sample after step(b).